Electrokinetic method for capturing and bioassaying airborne assayable pathogenic agents

ABSTRACT

Electrokinetic methods are described with the purpose of collecting assayable agents directed by creation of an electrokinetic potential well. Environmental agents such as biowarfare agents, pathogens, allergens or pollutants are collected autonomously. The dielectric fluid medium, such as air, is sampled by electrokinetic propulsion. A further embodiment for collection of pathogen samples entails exposing the sample to an electric plasma in the neighborhood of a high voltage electrode or electrodes, further transported through a potential well created at a sample collection device conductive liquids, such as oils may be sampled for the presence of contaminants, contaminating organisms or bio-degrading organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/955,150filed Nov. 30, 2010.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

MICROFICHE/COPYRIGHT REFERENCE

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the collection of and sampling ofassayable agents in a dielectric medium. This includes, but is notlimited to, sampling air for agents whose presence or absence isdeterminable by bio-specific assays. The field includes sampling of airfor biological agents, direction to, and deposition on, a collectionmeans for an assay device. The agent-specific assays may includeimmunoassays, nucleic acid hybridization assays, or any other assaysentailing ligand—antiligand interactions. Assays may include, but arenot limited to, detection means which are colorometric, fluorescent,turbidimetric, electrochemical or voltammetric. Agents assayed include,but are not limited to, bio-warfare agents, pathogens, allergens orpollutants. Pathogens include screening for infectious airborne agentssuch as anthrax or tuberculosis organisms. Further dielectric media mayinclude sampling of dielectric fluid medium such as oil for the foodindustry, or petrochemical and industrial oil.

2. Description of the Prior Art

In the prior art, there exist many examples of collection of agents fromthe air for bioassay. For example, the following publications describevarious methods of allergen, pathogen and toxin collection for assay:

Yao et al (2009) in Aerosol Science volume 40, pages 492-502

Noss et al (2008) in Applied and Environmental Microbiology, volume 74,pages 5621-5627

King et al (2007) in Journal of Allergy and Clinical Immunology, volume120, pages 1126-31

Earle et al (2007) in Journal of Allergy and Clinical Immunology, volume119, pages 428-433

Peters et al (2007) in Journal of Urban Health: Bulletin of the New YorkAcademy of Medicine, volume 84, pages 185-197

Yao and Mainelis (2006) in Journal of Aerosol Science, volume 37, pages513-527

Platts-Mills et al (2005) in Journal of Allergy and Clinical Immunology,volume 116, pages 384-389

Sercombe et al (2004) in Allergy, volume 60, pages 515-520

Custis et al (2003) in Clinical and Experimental Allergy, volume 33,pages 986-991

Poizius et al (2002) in Allergy, volume 57, pages 143-145

Tsay et al (2002) in Clinical and Experimental Allergy, volume 32, pages1596-1601.

Parvaneh et al (2000) in Allergy, volume 55, pages 1148-1154

McNerney et al (2010) in BMC Infectious Diseases, volume 10, pages161-166 and device in U.S. Pat. No. 7,384,793

Other known methods of sample collection include trapping of volatileorganic compounds (VOC) on activated carbon, de-sorption and analysis bymass spectrometry. See Phillips et al (2010) in Tuberculosis, volume 90,pages 145-151 and references therein. VOC's are not consideredencompassed by the present invention since the assays are strictlychemical in nature, and are not bio-specific as defined here. Bybio-specific is meant assays wherein the result is determined by abiological specificity such as nucleic acid specificity, antibodyspecificity, receptor-ligand specificity and the like. While diagnosticspecificity may be achieved by VOC analysis, this is inferred bypresence and amount of groups of defined organic compounds.

The foregoing prior art publications describe “dry” methods usingpumping and filtration, wiping, passive deposition, electrokinetictransport etc; usually followed by an extraction step and application ofthe extract to an assay.

Methods for collection in a liquid stream have been described in thepatent literature:

Yuan and Lin in US Patent Application 2008/0047429A1

Saski et al in U.S. Pat. No. 6,484,594 issued in 2002.

While efficiently collecting agents from the air, such liquid streamingsystems inevitably result in high dilution of the sample. There is aconsequent trade-off in sensitivity unless the agents arere-concentrated.

Northrup et al in U.S. Pat. Nos. 7,705,739 and 7,633,606 describe anautonomously running system for air sampling and determination ofairborne substances therein. They do not specify the exact method of airsampling, nor detail how it is transferred to an assay system.

There exist numerous commercially available systems for air purificationbased on filtration or electrostatic precipitation. For a generaldescription see the Environmental Protection Agency article “Guide toAir Cleaners in the Home”, U.S. EPA/OAR/ORIA/Indoor EnvironmentsDivision (MC-6609J) EPA 402-F-08-004, May 2008. Numerous commercialexamples of systems exist using either High Efficiency Particulate Air(HEPA) filters or electrostatic precipitation filters. Such systems arewidely used for removal of particulate matter or allergens from air,including as part of domestic heating, ventilation and air conditioning(HVAC) systems. HEPA filters have the advantage of removal of particlesdown to the micron size range, whereas electrostatic precipitationmethods have the advantage of entailing high volume flow with little orno pressure differential. See U.S. patent by Bourgeois, U.S. Pat. No.3,191,362 as a detailed example for the technical specification of anelectrostatic precipitation system. While efficiently removing agentsfrom the air, such air purification systems do not lend themselves tocollection of samples for analysis.

Electrokinetic-based air cleaning systems have been developed andformerly commercialized by the company Sharper Image (but nowdiscontinued) under the trade name Ionic Breeze. The originalelectrokinetic principle was enunciated by Brown in U.S. Pat. No.2,949,550. This was further improved by Lee in U.S. Pat. No. 4,789,801for improving airflow and minimizing ozone generation. Furtherimprovements for the commercially available system are described in USpatents by Taylor and Lee, U.S. Pat. No. 6,958,134; Reeves et al, U.S.Pat. No. 7,056,370; Botvinnik, U.S. Pat. No. 7,077,890; Lau et al, U.S.Pat. No. 7,097,695; Taylor et al, U.S. Pat. No. 7,311,762. In theforegoing descriptions of devices using electrokinetic propulsion, acommon element is a high voltage electrode consisting of a wire. A verysteep voltage gradient is generated orthogonally to the wire because ofthe very small cross-sectional area of the wire. The high voltagegradient causes the creation of a plasma consisting of chargedparticles, and kinetic energy is imparted to the charged particles bythe high voltage gradient. The resulting net air flow is created byexchange of kinetic energy between charged and uncharged particles, andthe net air flow is directed by the juxtaposition of planar electrodeswhich are at zero or opposite sign voltage to that of the wireelectrode. Charged particles are electrostatically precipitated on tothe planar electrodes, which may periodically be removed for cleaning.This body of work is directed toward air purification, not samplecollection. However, as first described by Custis et al (2003), theIonic Breeze device has been adapted for sample collection for allergenanalysis by wiping down the electrodes with a paper tissue. Theallergens were extracted from the tissue and subject to an immuno-assay.The Ionic Breeze was also used in the works of Peters et al (2007) andPlatts-Mills et al (2005) for allergen collection for immunoassayanalysis. Earlier, Parvaneh et al (2000) described an ionizer devicewith a “metal cup having a conductive surface as a collector plate”,from which allergens are extracted for assay. It is not evident how thesample is collected on the inside of a metal cup and does not adhere tothe entire surface. The device was made by Airpoint AB, Stockholm,Sweden. However, there is no public information concerning themanufacture or sale of such a product by Airpoint AB, there isinsufficient information for one skilled in the art to be able tounderstand the details of the device, and no similar device was used bythe same authors in subsequent publications on environmental allergendetection. There is no mention of focusing of the sample into apotential well created by a voltage gradient.

Yao et al (2009) and Yao and Mainelis (2006) have described methods forcollection of bio-assayable agents on to an assay means or device. Yaoand Manielis (2006) describe blocks of agar gel in electrical contactwith planar electrodes, and Yao et al (2009) describe a microtiter plateinterposed between planar electrodes. Both of these works describe aflow of air driven by a pump, and electrostatically precipitating theagents to be analyzed on to the assay means. The electrodes and the agarblocks have substantially the same area in these works.

McNerney et al (2010) describe a breathalyzer device, where theindividual breathes or coughs into a breathing tube, the sample collectson the internal surface of a tube, is scraped with a plunger on to anoptical biosensor, an immunological binding reaction is performed andthe biosensor utilizes an evanescent wave illumination system todetermine the presence or absence of M. tuberculosis by scattered light.

None of the above methods consider the use of an electric field gradientforming a potential well to focus the agents on to a collection meansfor an assay device.

SUMMARY OF THE INVENTION

The present invention encompasses the use of an electrode or electrodesto create a potential well that will draw charged particles out of aflowing dielectric fluid stream and focus them on to the collectionmeans of an assay device. The potential well serves to efficientlycapture the particles and enhance sensitivity by means of the focusingeffect on the collection means. The flowing air stream is created eitherelectrokinetically or mechanically. If not already electrically charged,charge is imparted to the agent to be analyzed by means of a highvoltage wire electrode arrangement and consequent plasma generation; theagent is focused on to the collection means of the assay device by thepotential well; and finally electrostatically precipitated thereon.

In one aspect of the invention, a device for collection of a sample froma dielectric fluid medium for a bio-specific assay device comprises anenclosure. Flow means direct fluid flow of the dielectric fluid mediumin the enclosure. One or more wire electrodes in the enclosure subjectdielectric fluid medium flowing in the enclosure to an ionizing plasma.Supporting means operatively associated with the enclosure support thebio-specific assay device. One or more capture electrodes are positionedproximate the supporting means to create a voltage potential wellwhereby charged particles thus generated within the dielectric fluidmedium, or pre-existing in said dielectric fluid medium, are propelledinto the supported bio-specific assay device therebyelectroprecipitating the charged particles on to a sample collectionregion of the bio-specific assay device.

Other objects, features, and advantages of the invention will becomeapparent from a review of the entire specification, including theappended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic cross-sectional representation of a prior artdevice for electrostatic precipitation as part of a HVAC system. Thishas been derived from technical literature from commercial suppliers;

FIG. 2 is a schematic cross-sectional representation of the Ionic Breezeprior art device, as published by Custis et al;

FIG. 3 is a cross-sectional representation of a device according to thepresent invention, wherein an collection means and assay device islocated adjacent to a plasma stream propelled by a fan;

FIG. 4 is a cross-sectional representation of the assay device showing adetail of FIG. 3;

FIG. 5 is a cross-sectional representation of a device according to thepresent invention, wherein the flow is achieved by electrokineticpropulsion, and the collection means of the assay device is adjacent toan aperture in a planar electrode of the electrokinetic propulsiondevice;

FIG. 6 represents a schematic cross section of an electrokinetic flowdevice wherein the assay device is a reel-to-reel collection device. Thecross section of this figure corresponds to section X . . . X of FIG. 5;

FIGS. 7a, b and c are various outputs from a computer simulation basedon the prior art Ionic Breeze device. In this and all following figures,a is the computer-aided design (CAD) input to simulation, b is theoutput represented by contour lines of voltage and c is the outputrepresented as a 3-dimensional plot with voltage as the third dimension;

FIGS. 8a, b and c are similar to the simulation of the prior art deviceof FIGS. 7 a, b and c, with a variation on the electrode geometry;

FIGS. 9a, b and c from a computer simulation of a further variation ofFIGS. 7a, b and c and 8 a, b and c showing a simplification of the priorart device with a reduction in the number of electrodes;

FIGS. 10a, b and c are outputs from the computer simulation of theforegoing figures showing a further simplification and beingrepresentative of the prior art device in U.S. Pat. No. 2,949,550;

FIGS. 11 a, b and c are outputs from the computer simulation of a priorare device corresponding to that of FIGS. 7a, b and c with thejuxtaposition of additional electrodes upstream, as described in U.S.Pat. No. 6,958,134;

FIGS. 12a, b and c are outputs of the computer simulation of the presentinvention with an electrode creating a potential well downstream to theplanar electrodes;

FIGS. 13a, b and c are outputs of the computer simulation of the presentinvention with an electrode creating a potential well adjacent to anaperture in a planar electrode;

FIGS. 14a, b and c are outputs of the computer simulation of the presentinvention similar to FIGS. 13a, b and c , but with an assay deviceinterposed in the potential well;

FIGS. 15a, b and c are outputs of the computer simulation of the presentinvention similar to FIGS. 14a, b and c , but with an assay deviceinterposed in the potential well, the assay device having a differentdielectric constant from that in FIGS. 14a, b and c;

FIGS. 16a, b and c are outputs of the computer simulation of the presentinvention similar to FIGS. 13a, b and c , but with elements of assaydevice on both sides of electrode creating potential well;

FIGS. 17a, b and c are outputs of the computer simulation of the presentinvention similar to FIGS. 10a, b and c , but additionally with anelectrode creating a potential well downstream to the planar electrode;

FIGS. 18a, b and c are outputs of the computer simulation of the presentinvention similar to FIGS. 17a, b and c but with an assay deviceinterposed in the potential well;

FIGS. 19a, b and c are outputs of the computer simulation of the presentinvention, with electrodes angled so as to enhance the air flow into thepotential well;

FIGS. 20-25 are outputs of a higher level computer simulation programwhich models electrostatic fields in three dimensions;

FIGS. 20 a, b, c and d represent CAD outputs as various stereographicprojections of a device according to the present invention;

FIGS. 21 a, b, c, d, e, f, g and h are electric field representations insuccessive planes proceeding along one axis of the device of FIG. 20;

FIGS. 22 a, b c and d are electric field representations in successiveplanes along a second axis of the device of FIG. 20;

FIGS. 23 a, b, c, d, e, f are electric field representations insuccessive planes along a third axis of the device of FIG. 20;

FIGS. 24 a, b, c and d represent CAD outputs as various stereographicprojections of a further device according to the present invention;

FIGS. 25a and b are electric field representations in two planes alongone axis of the device of FIG. 24.

DETAILED DESCRIPTION OF THE INVENTION

In its simplest embodiment, the present invention comprises a series ofwires held at high voltage in a flowing stream of air generated by afan-like device and a collection means of the assay device exposed tothe stream, and having an electrode juxtaposed so as to attract chargedparticles from the flowing stream. The juxtaposed electrode creates apotential well that will cause the charged particles to beelectrostatically precipitated on a collection area of the assay device.Thus, FIG. 3 illustrates such a device in detail. The device comprises anon-conductive housing 35, an entrance grill 31 and exit grill 37. Apumping device, such as a fan 36, directs and pulls through a flowingairstream entering at 30 and exiting at 38. Wire electrodes 32, seen incross-section in FIG. 3, are held at high voltage, in the range ofkilovolts, and plasma generation results in charged particles 33 whichare attracted out of the flowing airstream via a potential well on to acapture means 42 of the assay device 43. Assay device 43 incorporates anelectrode 41 which is shown to be grounded with the common electricalsymbol for grounding. The assay device 43 is removably supported andclamped in an indented aperture 39 in the housing 35. For clarity, theassay device 43 is separately illustrated in FIG. 4. Thus, to perform anassay, the assay device is clamped in aperture 39 in the housing 35 ofthe electrokinetic device of the present invention, exposed to apredetermined voltage, flow rate and time, then removed.

One form of assay would be a lateral flow immunochromatographic device,in which case the assay is initiated by application of a suitablechromatographic transport facilitating fluid to the sample well 42 andreading the result after a predetermined time. Numerous other simpleassay systems can also be used, involving optical or electrochemicaldetection and determination of the presence or absence or amount of theagent to be assayed. An alternative to the lateral flow device would bean enzyme immunoassay, where the presence or amount of the substance tobe detected is determined from application of a chromogenic substrate toan enzyme bound in immunocomplex, and determination of the subsequentcolor reaction. Since the device of the present invention performs in adielectric medium, conductive aqueous fluids would normally need to beadded to the various assay types to initiate a detection reaction.

The understanding of the present invention is facilitated by theillustrations of prior art devices. Further, because the presentinvention can be fabricated by simple modifications of prior artdevices, figures including drawings of several prior art devices havebeen included. FIG. 1 has been derived from various technicalspecifications of HVAC systems for domestic housing. Air flow is drivenby a fan 16, provide inlet flow 11 and outlet flow 18, entering via agrill 12, a coarse pre-filter 13 and the high voltage wire electrodes,grounded electrodes 15, upon which charged particles areelectrostatically precipitated, and exit grill 17. Prefilters are notnormally required in the function of the present invention.

In a further embodiment of the present invention, FIG. 5 shows a devicewith the flow of the dielectric fluid propelled by electrokinetic means.The device consists of a non-conductive housing 56, and entrance grillsand exits grills 52 and 57, respectively, for the inflowing andoutflowing dielectric fluid, 51 and 58, respectively. Wires 53, seen incross section in FIG. 5, are maintained at a positive high voltage, andelectrokinetic flow is directed by grounded planar electrodes 55 and 59.An aperture exists in the electrode 59, below which is located acollection means and assay device, removably supported in clamps 39 inthe housing 56. As in FIG. 4, the collection means incorporates anelectrode 41 which is small in dimensions compared with the groundedplanar electrodes 59. The electrode 41 in this case is held at a highnegative voltage in the kilovolt range. The planar electrodes 55 and 59cause the charged particles 54 to be first transported with the netfluid flow, but when they reach the potential well created by thenegative electrode 41, they are diverted from the stream on to thecollection area of the collection means 43. Thus, to perform an assay,the assay device is placed in a suitable aperture 39 in the housing 56of the electrokinetic device of the present invention, exposed topredetermined voltages of the wires 53 and the electrode 41 andpredetermined time, then removed. One form of assay would be a lateralflow immunochromatographic device, in which case the assay is initiatedby application of a suitable chromatographic transport facilitatingfluid to the sample well 42 and reading the result after a predeterminedtime. Numerous other simple assay systems can also be used, involvingoptical or electrochemical detection and determination of the agent tobe assayed. Since the device of the present invention performs in adielectric medium, conductive aqueous fluids would normally need to beadded to the various assay types to initiate a detection reaction.

In another embodiment of the present invention, an alternative means ofsample collection may be used to create a continuous record of the agentto be analyzed. FIG. 6 illustrates such an embodiment. The device ofFIG. 6 is comparable to the device of FIG. 5 in all respects except forthe omission of the assay device and replacement by a reel-to-reelsample collection means. The reel to reel device supports and moves thesample collection means orthogonally to the net flow of the dielectricfluid. Accordingly, the illustration of FIG. 6 represents a section X .. . X through the device of FIG. 5. The reels 61 and 62 rotate in thedirections indicated by arrows, transporting the sample collectionmeans, 62, through slots 64 in the housing 56. An electrode 63, mountedin the housing, is held at a negative voltage in the kilovolt range.Similarly to FIG. 5, charged particles will be swept out of the flowingstream by the potential well created by electrode 53, and deposited onthe sample collection means 62. Thus, to perform an assay, the wireelectrode 53 and the electrode 63, whose area is small compared with thegrounded planar electrodes 59, are set at predetermined voltages and thereel-to-reel transport device moves the sample collection means for apredetermined time. The sample collection device material may include apassive fibrous or membranous material, or an activated material thatwill capture the sample in place until the time of assay; or may be astructured material such as micro-pillar type, and may have embeddedcapture molecules, such as provide ligand-anti-ligand reactions. Uponcompletion of the predetermined time, the take-up reel 61 is removed andsubject to hydration prior to assay, in such a way that the capturedagent to be analyzed remains positioned on the capture means, either byactive or passive immobilization, or by capture via a ligand-anti-ligandinteraction. The assay is performed and the disposition of values alongthe length of the capture means provides a time record of the presenceor amount of the agent measured. The continuous record may becolorimetric, in which case the record is a visual display of thepresence or amount of the agent as a function of time. The continuousrecord may also be digital, in which the record can be presented as agraphic representation of the amount or presence of the agent as afunction of time.

FIG. 2 is reproduced from the publication of Custis et al (2003). Theprior art device of FIG. 2 comprises a housing 24, electrokineticallydriven air flow entering at 20 and exiting at 25, wire electrodes 21,and planar electrodes 23. Conjectural lines of constant voltage (voltagecontours) are shown as broken circles surrounding the electrodes 21, andconjectural particle movement in the airstream by arrows 22. Thisillustrates the particles impinging on and being electrostaticallyprecipitated on the planar electrodes, 23, which are removable. Samplesfor analysis are collected by wiping from the planar electrodes withtissue, extraction and application of the extract to an immunoassay,according to the procedure of Custis et al. The advantage of the presentinvention is that such separate wiping and extraction steps are notrequired. Further, while the contour lines of equal voltage in the priorart of Custis et al are conjectural, computer simulations are availablewhich facilitate the design of the devices of the current inventionwithout undue experimentation. Brown in FIG. 2 of U.S. Pat. No.2,949,550 also drew conjectural lines of voltage gradient. The voltagegradient will determine the force and direction experienced by a chargedparticle. Voltage gradients can be rigorously determined by computersimulation, eliminating undue experimentation.

The computer simulation of the devices of the present invention areperformed with the use of a software package provided by the companyField Precision LLC, PO Box 13595, Albuquerque, N. Mex. 87192, U.S.A.This software provided by Field Precisions LLC utilizes finite elementanalysis based on Coulomb's Law and Gauss's Law. The work is describedin “Field Solutions on Computers” (ISBN 0-8493-1668-5), author StanleyHumphries, published by CRC press. Description of the software and theconditions for purchase are provided by Field Precisions LLC. Theversion used here is the free students version, comprising the programMesh6.5 to design devices and EStat 6.0 to generate the output. Thedrawings of FIGS. 7a, b, and c to FIGS. 19a, b and c are generated withthis software package. FIGS. 20 a, b, c and d to 25 a and b aregenerated with the more advanced 3-dimensional programs, Geometer,Metamesh, HiPhi and PhiView. U.S. EPA/OAR/ORIA/Indoor EnvironmentsDivision (MC-6609J) EPA 402-F-08-004, May 2008

For further illustration of the use of the computer simulation, and todemonstrate how the present invention differs from the prior art,representations of prior art devices and arrangements are shown in FIGS.7a, b and c to FIGS. 11a, b and c . For better understanding of theapplication of the software package, a detailed description of theprocess is given for FIGS. 7a, b and c . A representation of the IonicBreeze configuration (FIG. 2) is created in the Mesh program in FIG. 7a. A bounding box of 4 units×4 units is defined, and within this box areplaced two points, 70, which represent the wire electrodes, and threelines, 71, which represent the planar electrodes. The symmetry isdefined as planar. This determines that all cross sections areequivalent extending in the third dimension out of the plane. Thisversion of the software performs the computation in two dimensions, thussimplifying the calculations. The Mesh program saves the file in a CADformat (suffix .DXF) and also converts to a script which is recognizedby the EStat program (suffix .MOU). The EStat then provides for theaddition of dimensions (units=inches), material properties such asdielectric constants (1 forair), and voltages (1000 for wire electrodes,0 for planar electrodes). With these parameters, a new file (suffix.EIN) is created. The mathematical solution of the simulation is thenperformed on the .EIN file, creating a file with the solution (suffix.EOU). Various graphical representations of the solution of the .EOUfile are then available. FIG. 7b shows the contour plot output, withcontour lines, lines of equal voltage, given a numerical value labelaccording to the voltage. FIG. 7c shows the surface plot format. Hereperspective drawing is used to express the voltage as a height in thethird dimension. The surface plot representation is particularly usefulas the steepness and direction of the slope in the surface representsvoltage gradient and direction. Thus, the surface plot represents theforce and direction vector to which a charged particle is subject. It isimmediately apparent from FIG. 7c that charged particles generated atthe wire electrodes, or pre-existing in the air, will be propelled downthe gradient into the three valleys and directed on to the surfaces ofthe planar electrodes.

The various configurations in the remaining FIGS. 8a, b, and c to FIGS.21a, b and c are all generated in this way.

For the establishment of design concepts for the present invention, theeffect of the thickness of the planar electrodes is shown in FIGS. 8a, band c . In FIGS. 7a, b and c the planar electrodes are represented ashaving zero thickness, whereas in FIGS. 8a, b and c they are representedas plates with a finite thickness of 1/20″. In all other respects, thesetwo sets of figures are identical. It can be readily seen that alteringfrom an infinitely thin electrode to one that has finite and practicalthickness has no impact on the resulting voltage gradients. The familyof U.S. Pat. Nos. 7,056,370, 7,097,695 and 7,311,762 teach theimprovement of reduction of thickness of the electrodes over the priorart, U.S. Pat. No. 4,789,801. However, further reduction of thicknesshas no benefit for the current invention.

For the purposes of the present invention, a somewhat simplerarrangement of electrodes would be advantageous for facilitating theplacement of a third electrode creating a potential well for the captureof the assayable agent on to the collection means of the assay device.Accordingly, the computer simulations in FIGS. 9a, b and c and 10 a, band c show the effect of successive reduction in the number ofelectrodes. FIG. 9a shows one high voltage wire electrode 90 and twoplate electrodes at 0 voltage, 91. The contour plot FIG. 9b and thesurface plot FIG. 9c show charged particles generated as plasma at thewire electrode 90, or pre-existing in the air, will be propelled downthe gradient into the two valleys and directed on to the surfaces of theplanar electrodes. Similarly, FIG. 10a shows a design with a single highvoltage wire electrode, 100 and a single plate electrode at zerovoltage, 101. The physical arrangement of FIG. 10a corresponds to theoriginal electrokinetic design of Brown in U.S. Pat. No. 2,949,550. Thecontour plot FIG. 10b and the surface plot FIG. 10c show chargedparticles generated as plasma at the wire electrode 100, or pre-existingin the air, will be propelled down the gradient into the valleys anddirected on to the surfaces of the planar electrode.

The design of FIG. 11a is identical with the design of FIG. 8a exceptfor the addition of two rod electrodes, 112, of 0.2 inches diameter anddisposed upstream of the wire electrodes 110 and the plate electrodes111. The electrodes 112 are held at 1000 volts, as are the wireelectrodes 110. The plots of FIGS. 11b and 11c show that the steepnessof the voltage gradient is compromised by the presence of the rodelectrodes 112, and the generation of plasma and electrokineticpropulsion would be reduced, although charges particles would still bedirected into the potential valleys adjacent to the planar electrodes111. It is to be emphasized that no focusing effect, in the sense usedin the current invention, is created. It is to be noted that Taylor andLee in the U.S. Pat. No. 6,958,134, teach that the placement of upstreamelectrodes serves to assist in the control of the flow of ionizedparticle. Nowhere do Taylor and Lee teach the use of an electrode ofsmall dimensions compared with the planar electrode as a means ofcreating a potential well to capture charged particles from a flowingfluid stream. FIGS. 11a, b and c shows that the focusing effect taughtby Taylor and Lee is distinct from the focusing as used in the presentinvention, as will be made clear from the embodiments of the presentinvention, which follow.

One embodiment of the present invention is shown in FIG. 12a . Thisconsists of two wire electrodes 120, three plate electrodes, 121 and acapture electrode, 123. This is comparable to the prior art device ofFIGS. 7a, b and c , but with the addition of capture electrode 123,according to the present invention. Electrodes 120 are at 1000 volts,the plates 121 at 0 volts and the capture electrode 123 is at −1000volts. The contour plot of FIG. 12b and the surface plot of FIG. 12cshow that electrokinetically driven charged particles generated by theplasma at the wire electrodes 120 will be driven to the potentialvalleys in the neighborhood of the plate electrodes 121, but thesevalleys are downward sloping as is clear in the surface plot of FIG. 12c. Consequently, the flow of charged particles will be propelled in thedirection of the capture electrode 123, and eventually will be trappedin the potential well created by the capture electrode 123. Note thatthe downward slope of the valleys in the neighborhood of the planarelectrodes 121 is less apparent in the contour plot FIG. 12b than in thesurface plot FIG. 12c . This is because the plot interval is adjustedfor clarity by the simulation program.

A more preferred embodiment of the current invention I illustrated inFIGS. 13a, b and c . The electrode arrangement is based on the prior artdevice of FIGS. 9a, b and c , with the following modifications accordingto the present invention. The electrode 132 is fabricated with a slot134, and juxtaposed with dimensions comparable to the slot 134 is thecapture electrode 133. See also the electrode arrangement of FIG. 5.This arrangement of creating a potential well off-set laterally to themain electrokinetic fluid flow, may, in certain designs according to thepresent invention, be more convenient for the insertion of a capturemeans and assay device than directly in the fluid stream. In spite ofthis off-set arrangement, the contour plot of FIG. 13b and the surfaceplot of FIG. 13c show the flow of charged particles will be propelled inthe direction of the capture electrode 133, and eventually will betrapped in the potential well created by the capture electrode 133.

For simplicity and ease of understanding, no capture means or assaydevice was included in FIGS. 13a, b and c . In FIGS. 14a, b and c , arepresentation of a capture means and/or assay device, 144, is included.This is placed between the slotted plate electrode 142 and the captureelectrode 143. A dielectric constant value of 2.0 for the capture meansand/or assay device is input into the computer simulation. Referencevalues for typical dielectric constants may be obtained from Handbook ofChemistry and Physics, CRC Press, Boca Raton, Fla., 91^(st) Edition,2010, section 13. The value 2.0 is that for dry paper. Comparing FIGS.13a, b and c with FIGS. 14a, b and c , the presence of paper as acapture means has no significant impact on the electric fielddistribution.

From the same web site, polystyrene resin has dielectric constant in therange 2.4-2.6. In order to cover the span of likely materials forcapture means and assay devices, a dielectric constant of 3.0 wasapplied to the capture means and/or assay device 154 in the computersimulation of FIGS. 15a, b and c . Again, no significant perturbation ofthe electric field distribution results, comparing FIGS. 13a, b and c ,14 a, b and c and 15 a, b and c. These foregoing simulations thus showthat there is great freedom in choice and disposition of capture meansand assay devices in designs of the present invention.

Capture means can be placed on both sides of the capture electrode, asillustrated by 164 and 165 in FIG. 16a . As in the preceding FIGS. 13a,b and c , 14 a, b and c and 15 a, b and c, there is no significantimpact on the electric field distribution.

A device according to the present invention based on the prior artdevice of FIGS. 10a, b and c is shown in FIGS. 17a, b and c . Thisconsists of a single wire electrode 170, a single planar electrode 171and, additionally, a capture electrode 172, situated downstream of theplanar electrode 171. This arrangement may be designed to capturecharged particles from the fluid flow by concentrating the stream on acenter line with the capture electrode centrally placed. Further, thedevice according to the resent invention in FIGS. 18a, b and c shows theadditional placing of a capture means, 183, of dielectric constant 2.0,between the planar electrode 181 and the capture electrode 182.

A preferred design according to the present invention is shown in FIGS.19a, b and c . Here three wire electrodes 190, and three planarelectrodes 191, are disposed at angles so as to maximize the fluid flowto converge on the capture electrode, 192, thus optimizing thecombination of fluid flow and electrokinetically directed flow in to thepotential well created by the capture electrode.

The foregoing computer simulation package is adequate to describe theprior art devices where all sections are equivalent for planes extendinginto a third dimension. However, it would be desirable to create devicesthat focus a charged particle stream in three dimensions, thus providinga true focusing effect. For this purpose, a higher level softwarepackage from Field Precision LLC is used. This package rigorously solvesthe same basic physical equations in three dimensional space by the samemethod of finite element analysis. The program is executed in threestages. The program Geometer has three-dimensional CAD features and isused for the creation of the initial design and visualization, forexample, by creation of stereographic diagrams. The program Metameshtakes the output from Geometer and creates the mesh for finite elementanalysis, and also inputs dimensions and various electrical and physicalproperties of the components. Hiphi solves the equations for the filescreated by Metamesh and Phiview performs further optional calculationsand provides for a variety of options for representing the output. Thus,the device created in Geometer is displayed in FIGS. 20a, b c and d.Here, the prior art device of FIGS. 9a, b and c is provided with anadditional capture electrode according to the present invention. Thewire electrode 200 is 10 inches in length, the plate electrodes 201 are10×10 inch squares and the capture electrode 202 is 0.5×0.5 inches.Plate electrodes 201 and capture electrode 202 are 0.1 inch thick. FIG.20a is a general stereographic view showing the orientations of the x, yand z axes. FIG. 20b is a view of the device looking down the x-axis,FIG. 20c is a view of the device looking down the y-axis and FIG. 20d isa view of the device looking down the z-axis. The definition of the axesand the orientation of the parts relative to these axes is important forthe understanding of FIGS. 21-23 since these represent successive planesprogressing through the device along the three axes. The deviceaccording to the current invention in FIGS. 20-23 is provided with avoltage of 1000 at the wire electrode 200, 0 volts at the plateelectrodes 201 and −1000 volts at the capture electrode 202. The Phiviewprogram can represent innumerable planes along each of the three axes,but for the purposes of illustration, only those planes which lie atcritical junctures in the device are represented here. Thus, FIG. 21a isin the y-z plane at the position X=−4.95

The position of the plane is indicated on the vertical axis of eachfigure. The contour lines of constant voltage are at approximately 100volt intervals. The density of the contours is an indication of thefield strength and hence the force applied to charged particles. Thearrows are vectors representing field direction in each cell for whichthere has been a calculation. Thus, in FIG. 21a there is a moderateforce field propelling charge particles away from the center line. Notethat this is only the component of the vector in the Y-Z plane, andhere, as everywhere else, the final direction is the result of vectorsin all three dimensions. The successive FIGS. 21b-21h then stepsuccessively through the entire device. FIG. 21b cuts through the planein which the wire electrode 200 lies, and shows very high field strengthpropagating out from the wire. Next, FIGS. 21c-e cut throughorthogonally to the planar electrodes at the extremities and at thecenter. The field intensity is relatively low in this region, being lessthan 10 volts per inch, as indicated by one or less contour lines. FIG.21f falls intermediate between the planar electrodes and showsincreasing voltage gradient in the direction of the center of thesection. FIG. 21g shows a section through the capture electrode andshows extremely high voltage gradient forming the potential well.Finally, the plane at x=5 inches shows moderating field strength, butcontinued direction of vectors to the center line. Surprisingly, anycharged particles exiting the device will be swept into the center ofthe y-z plane, and from FIGS. 22a and 23a , back into the potential wellalong the X axis.

FIGS. 22a-d show successive x-z planes progressing from the originoutward along the y axis. No sections for negative values of y are shownsince the device is symmetrical around the origin of the y-axis. Asimilar consideration holds for FIGS. 23a-f along the z-axis.

FIG. 22a confirms the findings of the two dimensional analysis softwarepackage, with the exception that out of the neighborhood of the centerline of FIG. 22a , vectors direct the stream away from the plateelectrodes, or downstream. Further, every section of FIG. 23 shows thex-y components of the vectors pointing downstream. Hence, theelectroprecipitation on the plate electrodes will be minimal. FIG. 23ashows the section including the wire electrode in the x-y plane, andincluding the capture electrode. In this section, the forces propellingcharged particles from the wire electrode into the potential well of thecapture electrode are apparent. FIG. 23b is a section of the x-y planejust proximal to the capture electrode, and then 23 c, 23 d and 23 eproximal to, cutting through and distal to the plate electrode, which isvisible in FIG. 23 d.

A further embodiment of the present invention is illustrated instereographic projections in FIGS. 24a, b and c generated in theGeometer program. This embodiment is intended to function as abreathalyzer device for breath-borne pathogens such as Mycobacteriumtuberculosis (M.tuberculosis). This can be implemented using a structuresimilar to FIG. 3 but eliminating the fan 36. Instead, the user blowsinto the entrance grill 31. The entrance grill 31 directs breathed airflow in the enclosure. FIG. 24a represents a general perspective view ofthe electrode arrangement, showing all three X, Y and Z axes. FIG. 24bis a view looking down the Y-axis, FIG. 24c is a view looking down theX-axis and FIG. 24d is a view looking down the Z-axis. The FIG. 24 showsonly the arrangement of the electrodes, and, for ease of understanding,the supporting structures which are made of materials in a range ofdielectric constants that do not influence the electric field, areomitted. The device includes 4 wire electrodes for generating plasma andtwo capture electrodes which may be used for collection means for twodifferent assay types. Thus, capture electrode 241 may be used for anoptical sensor device utilizing an immunoassay (immunosensor), asdescribed in detail in U.S. Pat. No. 7,384,793, while capture electrode242 may be used as a capture device for the nucleic acid polymerasechain reaction amplification based system Xpert MTB/RIF as described inBlakemore et al (2010) in Journal of Clinical Microbiology, volume 48,pages 2495-2501, Helb et al (2010) in Journal of Clinical Microbiology,volume 48, pages 229-237, and references therein. In a first phase, thetarget nucleic acid of the sample nucleic is recognized by ahybridization reaction and subsequently detected by real time polymerasechain reaction. An examples of immunosensor devices that may be used inconjunction with capture electrode 241 is where a fluorescent signalscattered from an immunocomplex at an immunosensor surface, illuminatedby an evanescent wave, is a measure of the substance to be analyzed. Theperformance of this embodiment is processed with MetaMesh and resultsgenerated with HiPhi. FIGS. 25a and b represent two views created fromPhiView. These two views selected from the complete three dimensionalanalysis are sufficiently representative to demonstrate the performance.FIG. 25a is a pseudo-3D contour plot showing the electric fielddistribution in an X-Y plane intersecting the origin of the Z-axis. TheZ-axis is not a physical Z-axis but represents the range 0-1000 volts.It can be seen from FIGS. 24 a, b, c and d that this plane willintersect all four wire electrodes and midway between the two captureelectrodes. It thus shows the formation of potential peaks at the wireelectrodes for the generation of plasma and a potential well in theneighborhood of the capture electrodes, which will serve to capture andelectro precipitate charged particles. A further contour plot in FIG.25b is the voltage distribution in a parallel plane displaced 1 inch outon the physical Z-axis. This plane skirts the extremity of the wireelectrodes, which are 2 inches in length. Here, too, can be seen thepotential peaks at the wire electrodes and the residual potential wellthat is here 0.5 inches beyond the extremity of the capture electrode.The ability to incorporate two capture electrodes in this case enhancesthe sensitivity of the assay by providing for two entirely differentassay systems for the same analyte. A further improvement is for theelectrode 242 to be replaced by a wire mesh of the same dimensions. Awire mesh electrode has the advantage of creating even greater localizedvoltage gradients in close proximity to the wires, and thus enhance thecapture effect. Following sample collection, the wire mesh electrode isremoved, immersed in 2 ml of the NaOH-isopropanol sample treatmentreagent, shaken for 5 seconds, incubated at room temperature for 15minutes, shaken again, and transferred to the Xpert MTB/RIF cartridgeand subject to the standard procedure for that assay device. TheNaOH-isopropanol reagent is provided by Cepheid Inc, the manufacturer ofthe Xpert MTB/RIF assay device.

Further multiplex capability can be attained by the use of amultiplicity of capture electrodes. While FIGS. 24a, b and c and 25 a, band c show the disposition of electrodes in the breathalyzer device,further details of mouthpiece, housing, and interface with an assaydevice are described in the specifications of U.S. Pat. No. 7,384,793 aswell as collection means and assay device commercialized by RapidBiosensor Systems Limited, Babraham, Cambridge, UK. A tubular orelliptical section housing can be constructed to accommodate themouthpiece, with entrance diameter optimized to match the dimensions ofthe wire electrodes and the exit diameter optimized to match thedimensions of the capture electrodes. The breath will then have maximumcontact with wire electrodes, and exit flow can be concentrated over thecapture electrodes.

It is apparent that the software packages provided by Field PrecisionLLC are useful for achieving optimal designs without undueexperimentation. Such programs have been under development for severalyears. P. L. Levin et al (“A Unified Boundary-element Finite-elementPackage” in IEEETransactions on Electrical Insulation 1993, volume 28,pages 161-167) made such a package available. Examples of application ofsuch software packages for the design of electrostatic precipitationdevices are given by S. Vlad (“Numerical Computation of ConductingParticle Trajectories in Plate-type Electrostatic Separators” in IEEETransactions on Industry Applications 2003, volume 39, pages 66-71) andby A. Bendoaoud et al (“Experimental Study of Corona Discharge Generatedin a Modified Wire-plate Electrode Configuration for ElectrostaticProcess Applications” in IEEE Transactions on Industry Applications2010, volume 46, pages 666-671). Optimization of the designs of thepresent invention is not limited to the software packages provided byField Precision LLC.

A key element of the present invention is the provision of a potentialwell that will act as a trap for charged particles of interest in aflowing fluid stream. It is possible to design innumerable deviceswithin the scope of this invention, and the configuration shown in theillustrations of this document are intended to be exemplary only. It issurprising that creation of a potential well provides a universal andefficient trap for charged particles and provides for seamless transferon to a measuring or detection device. The sensitivity of themeasurement of the detection or detection device is considerablyenhanced by the ability to sample large volumes of fluid and toconcentrate the charged particles on to a small area of a detectiondevice. Because the properties, disposition and dimensions ofnon-conducting materials do not significantly affect the voltage fielddistribution, there are unlimited possibilities for the design andfabrication of devices for practical applications, using, for exampleany of a wide range of plastic or polymeric non-conducting materials.

In the devices described in the foregoing, the area of the captureelectrode is small compared with other electrodes in the system, thusproviding a large voltage gradient. In the examples, typical ratios ofareas of capture electrodes are 20:1. Depending on the construction ofthe specific device, this ratio may vary in the range 5:1 to 1000:1 oreven greater, limited only by the performance requirements of thespecific system. The capture electrode is usually in the form of arectangular plate, but may also take the form of a metal grid or mesh.In the case of a multiplicity of wire electrodes for generating plasma,these are usually arrayed as parallel wires, but may also be arranged asa rectangular grid, depending on the requirements or constraints of aspecific design. The only constraint is that the geometry of the captureelectrode may not be such as to create a potential well with gradient sosteep as to initiate plasma generation, and generate charged particlesthat will be launched out of the potential well. The capture electrodedimension must be sufficiently small compared with the wire electrodesthat an advantageous focusing effect will take place. A rule of thumbmay be the use of the ratio of the longest dimension of the captureelectrodes to the sum of the lengths of the wire electrodes. A practicaluseful range may be with a lower limit of 1:5, below which a usefulconcentration of the charged particles may not take place. Smaller orlarger ranges may be useful for specific design requirements.

On the contrary, the wire electrodes must be of dimensions small enoughthat they will create a potential gradient sufficient to cause thegeneration of plasma. The wire electrodes advantageously do not exceed1.0 mm in diameter and in one embodiment may have a diameter ofapproximately 0.1 mm. However, the geometry of the wires may be variedand may also take the form of spikes with pointed tips. In this case,the pointed tip may give rise to a local potential gradient high enoughto give rise to the formation of charged plasma.

The voltages applied must be sufficiently large to create the conditionsfor the functioning of the invention, but voltages can be varied tooptimize the performance. The voltage values may be positive or negativeat either the wire electrodes or the capture electrodes. Forfunctioning, only relative voltages are important, so that any electrodemay also be set at ground or low voltage, for example, for safetyreasons.

For reduction to practice, the devices of the current invention can befabricated from simple modifications of existing devices. Thus, all thespecifications for details of hardware, electronic control, aestheticconsiderations, dimensions, portability, power supply from ac mains orbattery, are all described in detail in the prior art references givenin this document, and so need no further elaboration here.

Further applications to capture of entities to be assayed in dielectricmedia other than air can be created using the same principles asenunciated throughout this document. The dielectric fluid medium mayfurther include non-conductive liquids, such as oils. Oils may besampled for the presence of contaminants, contaminating organisms orbio-degrading organisms.

What we claim is:
 1. A method for determination of a pathogen orpathogens in an airborne sample volume comprising: subjecting a wireelectrode to a sufficiently high relative voltage to generate anionizing plasma; exposing said sample volume to said plasma; positioninga capture electrode at a low or negative voltage relative to said wireelectrode, thereby generating a net air flow, to propel chargedparticles generated by the ionizing plasma on to the capture electrode;running said propulsion for a time suitable for air flow of said samplevolume; and performing a biospecific assay to determine presence oramount of said pathogen in said sample volume.
 2. The method of claim 1further comprising a non-conductive transport medium covering saidcapture electrode to capture the sample volume.
 3. The method of claim2, wherein said non-conductive transport medium is subject to anextraction procedure with an extraction liquid.
 4. The method of claim3, wherein said extraction liquid is an NaOH-isopropanol sampletreatment reagent.
 5. The method of claim 1, wherein said pathogen is apathogenic member of the genus bacillus.
 6. The method of claim 1,wherein said pathogen is Mycobacterium tuberculosis.
 7. The method ofclaim 1, wherein said pathogen is a pathogenic virus.
 8. The method ofclaim 1, wherein said capture electrode is subject to an extractionprocedure with an extraction liquid.
 9. The method of claim 8, whereinsaid extraction liquid is an NaOH-isopropanol sample treatment reagent.10. The method of claim 1, wherein a plurality of capture electrodes ata low or negative voltage are positioned relative to said wireelectrode.